The goal of this research is to develop novel, more effective therapies for the treatment of tuberculosis, including multidrug resistant tuberculosis and related atypical mycobacterial infections. Isoniaizid is one of the most widely used chemotherapeutics for the treatment of tuberculosis and the molecular target of isoniazid has been thought to lie in the biosynthetic pathways involved in cell wall construction. Isoniazid is a prodrug that requires activation by an endogenous mycobacterial enzyme, the catalase-peroxidase KatG, to a toxic metabolite which then reacts with its specific target with lethal consequences. The target of activated isoniazid is the enzymatic extension of a 26-carbon fatty acid to the corresponding 28-carbon 2- ketoacyl acyl carrier protein (ACP) by a betaketoacyl ACP synthase, KasA. Section scientists, in collaboration with Professor James Musser, formerly of the Baylor College of Medicine, now at the NIAID Rocky Mountain Laboratories, have worked to confirm the identity of the putative target and to identify mutations in the gene encoding KasA from recent clinical isolates of Mycobacterium tuberculosis. The development of an in vitro assay for extension through KasA activity has allowed us to confirm that the observed mutations confer drug resistance to this enzyme. Ongoing work with Dr.s Jacob?s and Sacchetini has been directed towards obtaining a high-resolution structure of the enzyme targets of isoniazid action, KasA and AcpM.Isoniazid is one member of a family of small-molecule ?pro-drugs? clinically used in the treatment of tuberculosis. Another member of this family with a related molecular target is ethionamide. Ethionamide is a second-line antitubercular used in much of the world only with isolates resistant to front-line antituberculars (isoniazid, rifampicin and pyrazinamide). Isoniazid resistant isolates are generally not cross-resistant to ethionamide but ethionamide resistant isolates are occasionally cross-resistant to isoniazid. In addition to ethionamide we are currently studying two other second line antituberculars that we believe have related modes of action, thiocarlide (isoxyl) and thiacetazone. All three of these molecules have thiocarbonyl moieties as critical components of their chemical structures. These functional groups are believed to be metabolized by sequential S-oxidations, first to the sulfoxide and then to a more highly reactive bisoxygenated species. To study this process we have chemically synthesized radiolabelled ethionamide and thiocarlide and characterized their metabolism by intact cells of Mycobacterium tuberculosis. We have demonstrated that the sulfoxide is a transient species, that in vitro has a lower MIC, and oxidation of this species results in the covalent incorporation of label into cellular material currently being characterized. We have selected mutants resistant to these drugs and shown that the enzyme mediating resistance contains a cytochrome P-450 cofactor which accelerates their decomposition. The common thiocarbonyl active moiety and its activation has very important clinical implications. To study these we initiated a collaboration with a clinician in Cape Town, South Africa, where thiacetazone is used as front-line therapy. In drug resistant patient isolates following treatment with thiacetazone we have now confirmed cross-resistance to the other thiocarbonyl containing drugs with which these patients have not been treated. In collaboration with scientists at Pharmacopeia, Inc., Princeton, N.J. operating under NIAID CRADA #AI-0095 Section scientists have been involved in a high-throughput screening program for the elucidation of novel antibiotics active against the KasA pathway. These studies have involved utilization of a luciferase transcriptional reporter fused to the promoter for the kasA gene operon. This promoter has been shown to be responsive to treatment of the bacteria with isoniazid. This assay has been formatted for 96 well plates and implemented under Biosafety Level 3 conditions at NIAID. Using this assay we have screened approximately 1.5 million discrete chemical substances for their ability to inhibit mycolic acid synthesis. Several sub-libraries of compounds with unrelated chemical structures have been shown to be potent inhibitors of this crucial biosynthetic function. This screening program is entering a second phase designed to identify optimal binding inhibitors with novel bacteriocidal activities. The number of initial lead molecules compares very favorably with related screening projects being run at Pharmacopeia and scientists there are enthusiastic about the project outcome.